The goal of this work is to determine how cells die when their death is "programmed"; examples of such death include the involution of hormone-dependent tissues, death of cells that produces the final shape of developing organs, and possibly senescence. Within the immune system the death of thymus cells; of T cells when their growth factor IL-2 is removed; of irradiated lymphocytes; and of the targets of killer cells may all be programmed in that they all exhibit the same early event: the fragmentation of DNA into nucleosome-sized subunits. This fragmentation is the result of the action of an endogenous endonuclease. It will be isolated by biochemical fractionation techniques and monoclonal antibodies to it will be prepared. These will be used to screen a variety of cell types, both normal and those in which the death program has been induced. Besides the endonuclease, other proteins are involved in death; these have not yet been identified. DNA complementary to messenger RNA characteristic of dying cells will be isolated by subtractive hybridization, and used to probe a cDNA library made from dying cells. The selected clones will be used to identify those mRNAs specifically expressed in a wide range of dying cells, the "death genes". The appropriate cDNAs will then be sequenced, peptides corresponding to those sequences prepared, and monoclonal antibodies raised against them. These antibodies will be used to isolate the "death proteins", whose function will then be determined.